Herbal pharmaceutical compositions for prophylaxis and/or treatment of cardiovascular diseases and the method of preparing the same

ABSTRACT

The present invention provides an herbal pharmaceutical compositions comprising the root of scutellaria, the rhizome of coptis, the root and rhizome of rhubarb, and the dry powders of the root of ginseng (or American ginseng) or the rhizome of ginger. The herbal pharmaceutical compositions are effective in preventing patients from developing or treating patients with cardiovascular diseases, which include, but are not limited to, hypertension, coronary heart disease, cerebrovascular disease, peripheral vascular disease, heart failure, rheumatic heart disease, congenital heart disease, and cardiomyopathies. The present invention also provides methods for preparing and using the herbal pharmaceutical compositions.

RELATED APPLICATIONS

The present application is a Divisional Application of U.S. patentapplication Ser. No. 10/164,568, filed on Jun. 10, 2002 and now issuedas U.S. Pat. No. 6,793,944, which in turn claims the priority toTaiwanese application number 90131897, filed on Dec. 21, 2001, which areherein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to herbal pharmaceutical compositionswhich contain the root of scutellaria (Radix Scutellariae), the rhizomeof coptis (Rhizoma Coptidis), the root and rhizome of rhubarb (Radix etRhizoma Rhei), and the root of ginseng (Radix Ginseng) or Americanginseng (Radix Panacis Quinquefolii) for prophylaxis or treatment ofcardiovascular diseases. Optionally, the root of ginseng or Americanginseng can be replaced with the rhizome of ginger (Rhizoma Zingiberis).The herbs can be prepared as dry powders or extracts. The presentinvention also relates to the methods of preparing and using the herbalpharmaceutical compositions.

BACKGROUND OF THE INVENTION

Based on data from the World Health Organization (WHO), cardiovasculardiseases contribute to a third of global deaths in 1999 and areestimated to be the leading cause of death in developing countries by2010. Cardiovascular diseases are the name for a group of disorders inthe heart and blood vessels, including, but not limited to, hypertension(high blood pressure), coronary heart disease (heart attack),cerebrovascular disease (stroke), peripheral vascular disease, heartfailure, rheumatic heart disease, congenital heart disease, andcardiomyopathies.

Hypertension is by far the most prevalent cardiovascular disease. It isestimated that more than a third of Americans aged 45 or older have highblood pressure and, among them, more than 50% are aged 60 or older.Untreated hypertension can lead to serious and life-threateningcomplications, e.g., stroke, coronary heart disease, arteriosclerosis,atherosclerosis, heart failure, kidney failure and blindness.

As indicated in the United States Sixth Report of the Joint NationalCommittee (JNC VI) on High Blood Pressure, current treatment forhypertension includes diuretics, α-blockers, β-blockers, calcium channelblockers, ACE inhibitors, and angiotensin antagonists. These agents canbe used as monotherapy or in combination. However, most of these agentsameliorate the symptoms but not curing the diseases. These agents arealso frequently accompanied with side effects.

One of the major mechanisms for causing human hypertension is thedysfunction of endothelium. Endothelium is the layer of epithelial cellsthat lines the cavities of the heart and of the blood and lymph vessels.Its main role is to modulate both vascular tone and structure byproducing vasodilator and vasoconstrictor mediators.

When activated by specific agonists such as acetylcholine, endothelialcells produce nitric oxide (“NO”), a labile substance derived byL-arginine degradation through the activity of the endothelial NOsynthase (“eNOS”). NO is a powerful relaxing agent which also inhibitsplatelet aggregation and smooth muscle cell proliferation.

Under pathological conditions, such as hypertension or aging,agonist-induced stimulation of endothelium leads to activation of acyclooxygenase pathway and consequent production ofcyclooxygenase-dependent factors, including thromboxane A₂ orprostaglandin H₂, or free radicals (such as superoxide anions).Dysfunctional endothelium can also cause vascular damage, in particular,atherosclerosis.

There are two isoforms of cyclooxygenase, cyclooxygenase 1 and 2 (COX-1and COX-2), also referred to as prostaglandin endoperoxide synthase 1and 2, which are key enzymes in the conversion of arachidonic acid toprostaglandins, thromboxanes and other eicosanoids. It is believed thatCOX-1 and COX-2 have different physiologic functions due to strikingdifferences in their tissue expression and regulation. COX-1 is aconstitutive enzyme that is present at all times in the body and isresponsible for the production of cytoprotective prostaglandinsimportant for homeostatic functions, such as maintaining the integrityof the gastric mucosa, mediating normal platelet function, andregulating renal blood flow. In contrast, COX-2 is a rapidly inducibleform of cyclooxygenase that leads to the production of proinflammatoryprostaglandins. While COX-2 expression is highly restricted under basalconditions, it is dramatically up-regulated during inflammation. Theinvolvement of COX-2 and the elevated production of prostaglandins areassociated with a variety of diseases and disorders, such as brainischemia and cancers, as well as diseases and disorders in whichelevated levels of NO is present.

NO modulates the activity of COX-2 and participates in inflammatory andautoimmune-mediated tissue destruction. The effect of NO on COX-2 isdose-dependent. Low levels of NO activate COX-2. In contrast, largeamounts of NO produced by inducible nitric oxide synthase (“iNOS”) caninhibit the induction of COX-2 and suppress the formation of COX-2metabolites.

iNOS is expressed in the myocardium after myocardial infarction (MI) andin heart failure. Myocardium is the middle and thickest layer of theheart wall composes of cardiac muscle. Upregulation or overexpression ofiNOS is associated with mild inflammatory cell infiltrate, cardiacfibrosis, hypertrophy, and dilatation. Cardiac hypertrophy is asignificant risk factor for the development of congestive heart failure(CHF). Overexpression of iNOS results in overproduction of NO, causingmyocardial dysfunction and CHF.

CHF is a form of heart disease in which weakened heart function existswith concomitant edema. CHF has many different causes, includingnarrowing of the arteries supplying blood to the heart muscle (coronaryheart disease), prior heart attack (myocardial infarction) resulting inscar tissue large enough or located so to interfere with normalelectrocardiac function, high blood pressure, etc. CHF is one of themost serious cardiovascular diseases affecting adults. Over 4 millionpeople have CHF and the incidence is on the rise. The incidence of thisdisease or condition is increasing with the aging of the population andis currently the most common cause for hospital admission in theelderly. The total U.S. healthcare expenditure on CHF is over fivebillion dollars per year.

Atrial fibrillation (AF) is atrial arrhythmia characterized by rapidrandomized contractions of the atrial myocardium, causing a totallyirregular, often rapid ventricular rate. AF may persist due tostructural changes in the atria that are promoted by inflammation.C-reactive protein (CRP) is a marker of systemic inflammation whichpredicts cardiovascular events and stroke, a common sequela of AF. CRPalso induces adhesion molecule expression by endothelial cells.

While a panacea has been hunted for in western medicine for years,researchers turn to traditional Chinese herbal medicine for medicationsof various diseases. Chinese herbal medicine has existed and been usefor treating various diseases for thousands of years.

For example, San-Huang-Hsie-Hsin-Tang is an ancient herbal decoctionwhich was first described in Chin-Kuei-Yao-Lueh (translated into Englishas “the Prescriptions From the Golden Chamber”) for “purging fire andclearing the three torsos” and wherefore it is indicated forinsufficient cardiac “Chi,” hematemesis, and epistaxis. The decoction ismade of equal amounts of the root of scutellaria (Radix Scutellariae),the rhizome of coptis (Rhizoma Coptidis), and the root and rhizome ofrhubarb (Radix et Rhizoma Rhei). The decoction has a bitter taste andwith a cold nature. The decoction is intended for patients withcongestion, flush up, fidgets, shoulder stiffness, gastric obstructivedepression, constipation, and forceful pulse. However, the decoction iscontraindicated for not suitable for patients with symptoms of prolongedbleeding, marked anemia, and minute-weak pulses.

U.S. Pat. No. 5,443,839 discloses a composition havinganti-inflammatory, anti-allergic or anti-aging activity comprising,inter alia, an extract of Scutellaria. There is no indication that thecomposition is effective in treating cardiovascular disease andhypertension.

U.S. Pat. No. 6,274,177 discloses a method of preparing an extract fromZingiber officinale, which is potent in anti-inflammation andanti-platelet aggregation. There is no indication that the herbalcomposition is effective in treating cardiovascular disease andhypertension.

U.S. Pat. No. 6,340,480 discloses a composition and method for treatingcirculatory conditions including hypertension by promoting systemicvascular relaxation and dilation. The composition is a naturalcombination of L-arginine, ginseng, and Zizyphi fructus in an orally ortopically administered form. The combination works synergistically tosynthesize NO and thereby promote systemic vascular relaxation anddilation. The combined constituents may work to maintain a criticalthreshold level of NO in areas that cannot themselves produce it,thereby promoting systemic vascular relaxation and dilation in order toreduce hypertension. However, it is not clear whether the herbalcomposition is effective in treating cardiovascular disease.

In the invention to be presented in the following section, an herbalpharmaceutical composition is described. This herbal pharmaceuticalcomposition is effective in both prophylaxis and treatment ofcardiovascular diseases. The composition is also non-toxic and thus canbe used by patients in all ages and physical conditions, including theweek, the early and the debilitated.

In addition, the herbal pharmaceutical composition of the presentinvention provides multiple mechanisms of pharmacologically effects,including lowering and stabilization of blood pressure; inhibition ofexpression of iNOS; inhibition of COX-2 activity; reduction of bloodCRP; and reduction of blood cholesterol.

SUMMARY OF THE INVENTION

The present invention provides herbal pharmaceutical compositions whichcontain Radix scutellariae (root of scutellaria); Rhizoma Coptidis(rhizome of coptis); Radix et Rhizoma Rhei (root and rhizome ofrhubarb); and Radix Ginseng (root of ginseng) or Radix PanacisQuinquefolii (American ginseng). The preferred weight ratio of the rootof scutellaria, the rhizome of coptis, the root and rhizome of rhubarb,and the root of ginseng or American ginseng is about 1–2:1–2:1–2:1–2;and most favorably 1:1:1:1. Optionally, the root of ginseng or Americanginseng can be replaced with Rhizoma Zingiberis (rhizome of ginger). Thepreferred weight ratio of the root of scutellaria, the rhizome ofcoptis, the root and rhizome of rhubarb, and the rhizome of ginger isabout 1–2:1–2:1–2:1–2; and most favorably 1:1:1:1.

The herbs of the present invention can be prepared in the form of drypowders or extracts. The herbal pharmaceutical composition containingdry powders of the root of scutellaria, the rhizome of coptis, the rootand rhizome of rhubarb, and the root of ginseng or American ginseng (asshown in Example 1, infra) is pharmaceutically active and possesses theproperties of lowering and maintaining blood pressure as well astreating other cardiovascular diseases. However, the preferredpharmaceutical compositions of the present invention are the onescontaining a mixture of both herbal extracts and dry powders.Preferably, the root of scutellaria, the rhizome of coptis, and the rootand rhizome of rhubarb are prepared as extracts, where the activeingredients of the herbs are extracted by a solvent, which can be water,alcohol, or a mixture thereof. The preferred solvent for the root ofscutellaria and the rhizome of coptis is water. The preferred solventfor the root and rhizome of rhubarb is alcohol, most favorably 95%alcohol (in water).

The preferred form of ginseng/American ginseng or ginger used in theherbal pharmaceutical compositions is dry powders, which are prepared bycutting and grinding the herbs, followed by drying.

In addition to the herbs, the herbal pharmaceutical compositions of thepresent invention can contain a pharmaceutically acceptable excipientand/or carrier and be formulated in various dosage form such as granule,capsule, tablet, powder, and bolus, for orally administration. Thepreferred formulation is tablet.

The present invention provides a methods for preparing the herbalpharmaceutical compositions which contain a mixture of herbal extractsand dry powders as follows: (1) individually extracting the root ofscutellaria, the rhizoma of coptis, and the root and rhizome of rhubarbwith an appropriate solvent to form respective extracts of the herbs;(2) individually filtering the respective extracts of the herbs; (3)mixing the respectively filtered extracts of the herbs to form an herbalmixture; (4) condensing the herbal mixture to form an herbal paste; (5)grinding the root of ginseng or American ginseng to make dry powders ofginseng or American ginseng; (6) adding the dry powders of ginseng orAmerican ginseng to the herbal paste; and (7) drying the paste to formthe herbal pharmaceutical compositions.

Optionally, the root of ginseng or American ginseng can be replaced withRhizoma Zingiberis (rhizome of ginger).

The root of scutellaria and the rhizome of coptis are preferablyextracted by water. The root and rhizome of rhubarb can either beextracted by alcohol or by a mixture of alcohol and water, preferably95% alcohol in water.

The herbal pharmaceutical composition of the present invention hastherapeutic effect on cardiovascular diseases and can be used forprevention and/or treatment of cardiovascular diseases, including, butnot limited to, hypertension, coronary heart disease, cerebrovasculardisease, peripheral vascular disease, heart failure, rheumatic heartdisease, congenital heart disease, and cardiomyopathies.

Specifically, the herbal pharmaceutical composition of the presentinvention can stabilize and lower blood pressure, prevent damage toendothelial cell (e.g., by inhibiting iNOS activity, inhibiting COX-2activity, reducing blood CRP concentration, inhibiting smooth muscularcell proliferation, and reduce blood cholesterol level). The herbalpharmaceutical composition can be safely used by patients at any agesand physical conditions, including the weak, the elderly, and thedebilitated.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the anti-hypertensive effects of: (1) the root ofscutellaria alone (H1); (2) the root and rhizome of rhubarb alone (H3);(3) a combination of the root of scutellaria and the rhizome of coptis(H1+2); (4) the modified San-Huang-Hsie-Hsin-Tang decoction (M) as shownin COMPARATIVE EXAMPLE 1, infra; (5) STC (Example 3), infra; and (6) acombination of captopril and nifedipine (A) in rats. Captopril is an ACEinhibitor. Nifedipine is a calcium channel blocker. * indicatesstatistical significance.

FIGS. 2 a and 2 b show the effects of: (1) placebo control (C); (2) themodified San-Huang-Hsie-Hsin-Tang decoction as shown in COMPARATIVEEXAMPLE 2 (M), infra; (3) STC (Example 3, infra); and (4) thecombination of captopril and nifedipine (A); on mean arterial bloodpressure (maBP) and heart rate (HR) of rats, respectively, on the dayafter a completion of 2-week dose administration.

FIGS. 3 a and 3 b show the effects of STC (Example 3, infra) on heartrate (HR) and blood pressures, respectively, of rats, after the animalswere given a daily dose of 14.4 mg/kg for one week. SHR represents thebaseline control of the spontaneous hypertensive rats. WKY representsthe baseline control of the normal rats. STC were given to thespontaneous hypertensive rats. SYS, DIA, and maBP represent thesystolic, diastolic and mean arterial blood pressure, respectively.

FIGS. 4 a and 4 b compare the heart rates (HR), and the systolic (SYS),diastolic (DIA) and mean arterial blood pressures (maBP), respectively,in 8-week-old WKY rats (Young), 10-month-old WKY rats (Old) and10-month-old WKY rats treated with 48 mg/kg of the herbal pharmaceuticalcomposition of the present invention (STC).

FIG. 5 shows inhibition of nitrite formation in the mouse macrophagecell line RAW 264.7 by STC. The symbol, C, represents the untreatedcontrol mouse macrophage cells. LPS and STC represent the mousemacrophage cells treated with lipopoly-saccharide and the herbalpharmaceutical composition of the present invention, respectively.

FIG. 6 shows the inhibitory effects of the herbal pharmaceuticalcomposition of the present invention on PEG-2 formation by mousemacrophage cell line RAW 264.7. LPS and STC represent the mousemacrophage cells treated with lipopolysaccharide and 10 mg/mL of theherbal pharmaceutical composition of the present invention.

FIGS. 7 a and 7 b show inhibition on the protein syntheses of COX-2 andiNOS, respectively, by the herbal pharmaceutical composition of thepresent invention (STC). Column 1 presents the enzymes isolated from theheart of spontaneous hypertensive rats treated with 20 mg/kg oflipopolysaccharide. Column 2 presents the enzymes isolated from thelungs of spontaneous hypertensive rats treated with the herbalpharmaceutical composition of the present invention. Column 3 presentsthe enzymes isolated from the lungs of the control, untreatedspontaneous hypertensive rats.

FIGS. 8 a and 8 b show RNA expressions of β-actin and iNOS,respectively, in the lung of spontaneous hypertensive rats that weretreated with (1) the modified San-Huang-Hsie-Hsin-Tang decoction (M)(cDNA marker), (2) STC (Example 3, infra) (S), (3) control (C), and (4)a combination of captopril and nifedipine (A).

FIG. 9 shows the ELISA analysis of CRP in the blood of spontaneoushypertensive rats of the control group (C) and the group treated with 48mg/kg of STC (Example 3, infra) for two weeks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel herbal pharmaceutical compositionsfor preventing and treating cardiovascular diseases, which are suitablefor patients of all age groups and physical conditions, includingelderly and debilitated.

Recent progress in the scientific and medical understanding of thecardiovascular diseases provide more knowledge on the involvement ofendothelial damages, nitric oxide (NO), inflammation reactions andC-reactive protein (CRP) in these diseases. NO produced in theendothelium by the endothelial nitric oxide synthase (eNOS) is not onlya potent vasodilator but also inhibits platelet aggregation, smoothmuscle cell proliferation, monocyte adhesion and adhesion moleculeexpression, thus, maintain the integrity of the endothelial tissues.Production of cyclooxygenase (COX)-dependent factors, includingprostanoids and oxygen free radicals, may be the main cause forendothelial dysfunction. Dysfunctional endothelium can then be one ofthe main mechanisms causing vascular damage which can further lead tomore severe cardiovascular diseases. As inhibition of cyclooxygenase mayrestore NO-mediated vasodilation in essential hypertension,anti-inflammatory interventions may have therapeutic utility.

During diseased states, e.g., cardiac hypertrophy, myocardial infarction(MI), ischemia, myocarditis and septic shock, overexpression of theinducible nitric oxide synthase (iNOS) leads to increased production ofNO. The elevated NO levels can result in more severe complications,e.g., myocardial dysfunction, congestive heart failure and suddencardiac death.

During the inflammation process, an acute-phase reactant, C-reactiveprotein (CRP), is formed. CRP, frequently used as a systemicinflammation marker, promotes the expression of the adhesion moleculesand may plays a direct role in the pathogenesis of vascularinflammation, particularly atherosclerosis. CRP has been associated withvascular risk factors and with prevalent and incident atherothromboticcardiovascular diseases, i.e., coronary heart disease, stroke, andperipheral arterial disease.

A new approach for treatment of cardiovascular diseases should considerall aspects of the diseases. For example, in addition to theanti-hypertensive activity, the new medicine should not only be able toprotect healthy endothelium but also improve the functions ofdysfunctional endothelium.

The present invention provides herbal pharmaceutical compositions thathave the functions of reducing high blood pressure, maintaining normalblood pressure, improving and protecting cardiovascular endothelial cellfunctions, so as to treat and prevent cardiovascular diseases.

The herbal pharmaceutical compositions of the present invention containfour herbs: the root of scutellaria, the rhizome of coptis, the root andrhizome of rhubarb, and the root of ginseng or American ginseng.Optionally, the root of ginseng or American ginseng can be replaced withthe rhizome of ginger.

The herbs used in the compositions can be any variants of the herbsmentioned above. For example, the root of scutellaria has three closelyrelated variants, which are, Scutellaria baicalensis Georgi, Scutellariaviscidula Bge., and Scutellaria amoena C. H. Wright. The rhizome ofcoptis has four closely related variants, which are, Coptis chinensisFranch., Coptis deltoidea C. Y. Cheng et Hsiao, Coptis teetoides C.Y.Cheng, and Coptis omeiensis (Chen) C.Y. Cheng. The root and rhizome ofrhubarb has three closely related variants, which are Rheum palmatum L.,Rheum tanguticum Maxim., and Rheum officinale Baill.

The pharmaceutical effect of scutellaria is in the dried root, which hasthe pharmaceutical name of Radix Scutellariae. Scutellaria belongs tothe family of Labiatae. The herb is mainly produced in the provinces ofHebei, Shanxi, and inner Mongolia of China. The best harvest seasons forthe herb are in spring or autumn. The root of scutellaria is dried underthe sunlight, sliced, and used unprepared or stir-baked with wine orstir-baked to charcoal. The herb is bitter in flavor and cold inproperty. According to traditional Chinese medicine, the herb can beused to cure diseases in lung, gallbladder, stomach, and large intestinechannels. Specifically, the herb can be used to remove damp-heat,counteract toxicity, arrest bleeding, and prevent abortion in patients.

The root of scutellaria contains active ingredients which include, butare not limited to, baicalin, oroxylin A-glucuronide,wogonin-7-O-glucuronide, baicalein, wogonin, and oroxylin A. Baicalincan be used as a denominator for qualitative or quantitative control ofthe herb.

The pharmaceutical effect of coptis is in the dried rhizome of coptis,which has the pharmaceutical name of Rhizoma Coptidis. Coptis belongs tothe family of Ranunculaceae. It is mainly produced in the provinces ofSichuan, Hubei, and Yunnan of China. The preferred harvest season is inautumn. The rhizome of coptis is dried under the sunlight after therootlets and earth have been removed and used unprepared or stir-bakedwith ginger juice. The herb is bitter in flavor and cold in property.According to traditional Chinese medicine, the herb can be used to curediseases in the heart, stomach, liver, and large intestine channels.

The rhizome of coptis contains active ingredients, which include, butare not limited to, berberastine, columbamine, jatrorrhizine,epiberberine, coptisine, palmatine, and berberine. Berberine can be usedas a denominator for the qualitative or quantitative control of theherb.

The pharmaceutical effect of rhubarb is in the dried root and rhizome ofrhubarb, which has the pharmaceutical name of Radix et Rhizoma Rhei.Rhubarb belongs to the family of Polygonaceae. It is mainly produced inthe provinces of Qinghai and Sichuan of China. The root and rhizome ofrhubarb is dug in the late autumn when its stem and leaves begin towither, or in the early spring before the plant begins to sprout. Theharvested herb is dried and sliced. The root and rhizome of rhubarb canbe used unprepared, stir-baked with wine, or carbonized. It is bitter inflavor and cold in property. According to traditional Chinese medicine,the root and rhizome of rhubarb can cure diseases in spleen, largeintestine, liver, and heart channels.

The root and rhizome of rhubarb contains active ingredients, whichinclude, but are not limited to, sennoside B, sennoside A, aloe-emodin,rhein, emodin, and chrysophanol. Sennoside A and/or emodin can be usedas denominators for qualitative or quantitative control of the herb.

There are two kinds of ginseng, Radix Ginseng (root of ginseng) andRadix Panacis Quinquefolii (root of American ginseng), which can be usedin the herbal pharmaceutical composition of the present invention. RadixGinseng is Panax ginseng C. A. Mey. Radix Panacis Quinquefolii is Panaxquinquefollum L. Radix Ginseng belongs to the family of Araliaceae.Ginseng is mainly produced in the provinces of Jilin, Liaoning, andHeilongjiang of China. Ginseng produced in Fusong of Jilin isparticularly of good quality. The herb can also be cultivated, which iscalled “garden ginseng,” as opposed to “mountain ginseng” which refersto the ginseng found in the wild. Cultivated ginseng is harvested inautumn. The harvested ginseng is dried in the sun or roasted, which iscalled “sun-dried ginseng,” or dried after being steamed, which iscalled “red ginseng,” or soaked in syrup, which is known as“sugar-processed ginseng.” The fibrous rootlets are known as ginsengrootlets. Wild ginseng dried in the sun is known as sun-cured wildginseng. The herb is sliced for use. The herb has a sweet and slightlybitter flavor and is neutral in property. According to traditionalChinese medicine, ginseng is particularly good for curing diseases inspleen, lung, and heart channels.

The pharmaceutical effects of ginseng are in its dried root. Ginsengalso has effects on central nervous system. It enhances both stimulatoryand inhibitory processes in the central nervous system, therebyimproving the adaptability of nervous responses. Ginseng can also lowerserum glucose and cholesterol. It also shows therapeutic and preventiveeffect on peptic ulcer.

Radix Panacis Quinquefolii also known as American ginseng, belongs tothe family of Araliaceae. The medicinal effects of American ginseng isin the root. American ginseng can be found in northern United States andCanada. It has also been widely cultivated in France and northern China.The best harvest season for American ginseng is in autumn.

The active ingredients in the root of ginseng or American ginsenginclude, but are not limited to, ginsenoside Rg1, ginsenoside Re, andginsenoside Rb1, among which ginsenoside Rb1 can be used as adenominator for qualitative or quantitative control of the herb.

The pharmaceutical effect of ginger is in the dried rhizome of ginger,which has the pharmaceutical name of Rhizoma Zingiberis. Ginger belongsto the family of Zingiberaceae. Fresh rhizome or dried rhizome of gingercan be prepared by baking, roasting, and simmering. Ginger can be foundthroughout China, especially Sichuan, Guizhou, Guangdong. It isprimarily cultivated. The best season for harvest is before the wintersolstice. Ginger is acrid and has warm property. According totraditional Chinese medicine, ginger is particularly good for curingdiseases in spleen, lung, and stomach.

The active ingredients in the rhizome of ginger include, but are notlimited to, gingerol, shogaol, and zingerone, among which gingerol canbe used as a denominator for qualitative and quantitative control of theherb.

The pharmaceutical names, botanical or zoological names, family names,common descriptions, and major ingredients of the herbs used in thepresent invention are shown in Table 1.

TABLE 1 Herbs of the Present Pharmaceutical Composition PharmaceuticalBotanical Common Name Name Family Description Major Ingredients RadixScutellariae Scutellaria Labiatae scutellaria or baicalein, baicalin,wogonin, baicalensis scute wogonin-7-0-glucuronide, Georgi neobaicalein,oroxylin A glucuronide, camphesterol, β- sitosterol, benzoic acidRhizoma Coptidis Coptis chinensis Ranunculaceae coptis rhizomeberberine, coptisine, worenine, Franch., C. palmatine, columbamine,deltoidea C. Y. obacunone, obaculactone, Cheng, C. palmatine,jatrorrhizine, omeiensis magnoflorine, ferulic acid (Chen) C. Y. Cheng,or C. teetoides C. Y. Cheng Radix et Rhizoma Rheum polygonaceae rhubarbroot and derivatives of anthraquinone Rhei palmatum L. or rhizomeglycosides including R. tanguticum chrysophanol, emodin, aloe- Maxim. etReg. emodin, rhein, and physcion, (used in north rheum tannic acids,gallic acid, China) or R. catechin, tetrarin, glucogallin, officinaleBaill. cinnamic acid, rheosmin, fatty (used in south acids, calciumoxalate, glucose, China) fructose, sennoside A, B, and C RhizomaZingiber Zingiberaceae ginger, ginger gingerol, shogaol, and zingeroneZingiberis officinale rhizome Roscoe Radix Panacis Panax AraliaceaeAmerican saponins, panaquilon Quinquefolii quinquefolium L. ginsengRadix Ginseng Panax ginseng Araliaceae ginseng, red panaxatriol,panaxadiol, other (Rubra) C. A. Mey ginseng panoxisides, panoquilon,panaxin, ginsenin, α-panaxin, protopanaxadiol, protopanaxtriol,panacene, panaxynol, panaenic acid, panose, dammarane, glucose,fructose, maltose, sucrose, nicrotinic acid, riboflavin, thiamine

In the pharmaceutical compositions of the present invention, the root ofscutellaria, the rhizome of coptis, and the root and rhizome of rhubarbare best prepared by solvent extraction. The solvent can be water or anypharmaceutically acceptable organic solvent or a mixture of water andthe organic solvent. The organic solvent is preferred to be alcohol.

In the pharmaceutical composition of the present invention, the weightratio of the root of scutellaria, the rhizome of coptis, the root andrhizome of rhubarb, and the root of ginseng or American ginseng is about1–2:1–2:1–2:1–2, most favorably 1:1:1:1.

Optionally, the rhizome of ginger can replace the root of ginseng orAmerican ginseng.

The pharmaceutical compositions of the present invention can beformulated into tablet, bolus, powder, capsule, and granule by meanssuitable and known in the art and the pharmaceutical industry. Thepreferred formulation is tablet.

Quality Control of the Herbs

The present invention uses High Performance Liquid Chromatography (HPLC)to fingerprint each herb for the purpose of ensuring quality ofindividual herbal ingredients. The HPLC method and the test results ofthe herbal components of the present invention are described as follows:

A. Preparation of Herbal Extracts for HPLC

-   -   (1) 0.5 gram of the herbal component was precisely weighed and        placed in a 50-mL sample bottle.    -   (2) 20 mL of 70% methanol was added to the sample bottle of (1).    -   (3) The mixture of (2) was sonicated at room temperature for 15        minutes and further shaked in a 40° C. water bath at 160 rpm for        20 minutes; the sample was then sat for 30 minutes or more until        two layers of the solution was formed.    -   (4) The clear, upper layer of the solution was taken out and        passed through a 0.45 μm PVDF filter made by Whatman, England.    -   (5) About 20 μL of the filtered solution was injected into the        HPLC for quantitative analysis.        B. Instruments for HPLC Analyses

The instruments used include Waters 600E Pump, Waters 717PlusAutosampler, and Waters 996 Photodiode Array Detector.

C. HPLC Conditions and Results of Individual Herbs

1. Rhubarb

(a) HPLC Conditions:

-   -   Guard column: Lichrospher RP-18 endcapped        -   (5 μm, 4.0 ID×10 mm, Merck, German)    -   Column: Symmetry Shield RP 18        -   (5 μm, 4.6 ID×250 mm, Waters, USA)    -   Column temperature: 40° C.    -   Mobile phase: A: 0.5% acetic acid (CH₃COOH) in water        -   B: acetonitrile (CH₃CN)    -   Elution Gradients:

Time (minutes) A (%) B (%) Linearity 0 86 14 * 40 75 25 linear 60 55 45linear 70 55 45 linear 90 0 100 linear 100 86 14 linear

-   -   Flow rate: 0.85 mL/min    -   Detection wavelength: 270 nm

(b) Results:

-   -   The HPLC chromatogram of the root and rhizome of rhubarb        contains the indicative ingredients of sennoside B, sennoside A,        aloe-emodin, rhein, emodin, and chrysophanol. The retention        times and maximum absorption wavelengths of these ingredients        are shown in Table 2.

TABLE 2 Retention Times and Wavelengths of the Ingredients in RhubarbRetention Time Maximum absorption Compound (minutes) wavelength (λmax)Sennoside B (SB) ~38 268 nm Sennoside A (SA) ~46 269 nm Aloe-emodin (AL)~72 277 nm Rhein (RH) ~87 257 nm Emodin (EM) ~92.5 287 nm Chrysophenol(CH) ~94 256 nm2. Scutellaria

(a) HPLC Conditions:

-   -   Guard column: Lichrospher RP-18 endcapped        -   (5 μm, 4.0 ID×10 mm, Merck, German)    -   Column: Cosmosil 5C18-MS        -   (5 μm, 4.6 ID×250 mm, Nacalai tesque, Japan)    -   Column temperature: 35° C.    -   Mobile phase: A: 20 mM KH₂PO₄ and 0.01% H₃PO₄ in water        -   B: acetonitrile (CH₃CN)        -   C: water (H₂O)    -   Elution Gradients:

Time (minutes) A (%) B (%) C (%) Linearity 0 87 13 0 * 25 75 25 0 Linear40 65 35 0 Linear 55 0 75 25 Linear 60 87 13 0 Linear

-   -   Flow rate: 1.0 mL/min    -   Detection wavelength: 280 nm

(b) Results:

-   -   The HPLC chromatogram of the root of scutellaria contains the        indicative ingredients of baicalin, oroxylin A-glucuronide,        wogonin-7-O-glucuronide, baicalein, wogonin, and oroxylin A. The        retention times and maximum absorption wavelengths of these        ingredients are shown in Table 3.

TABLE 3 Retention Times and Wavelengths of the Ingredients inScutellaria Retention Time Maximum absorption Compound (minutes)wavelength (λ_(max)) Baicalin (BG) ~30 276 nm Oroxylin A-glucuronide(OG) ~36 269 nm Wogonin-7-O-glucuronide (WG) ~39 272 nm Baicalein (B)~51 275 nm Wogonin (W) ~56 274 nm Oroxylin A (O) ~57 269 nm3. Coptis

(a) HPLC Conditions

-   -   Guard column: Lichrospher RP-18 endcapped        -   (5 μm, 4.0 ID×10 mm, Merck, German)    -   Column: Cosmosil 5C18-MS        -   (5 μm, 4.6 ID×250 mm, Nacalai tesque, Japan)    -   Column temperature: 35° C.    -   Moble phase: A: buffered acetonitrile (The buffer contains 50 mM        of CH₃COONa, 2% CH₃COOH, and 5 mM C₁₂H₂₅OSO₃Na)        -   B:H₂O:CH₃CN:CH₃OH=10:45:45 (v/v)    -   Elution Gradients:

Time (minutes) A (%) B (%) Linearity 0 100 0 * 15 65 35 linear 30 65 35linear 40 100 0 linear

-   -   Flow rate: 0.85 mL/min    -   Detection wavelength: 270 nm

(b) Results:

-   -   The HPLC chromatogram of the rhizome of coptis contains the        indicative ingredients of berberastine, columbamine,        jatrorrhizine, epiberberine, coptisine, palmatine, and        berberine. The retention times and maximum absorption        wavelengths of these ingredients are shown in Table 4.

TABLE 4 Retention Times and Wavelengths of the Ingredients in CoptisRetention Time Maximum absorption Compound (minutes) wavelength (λmax)Berberastine (Berber) ~17 264 nm; 357 nm Columnbamine (Col) ~21 264 nm;345 nm Jatrorrhizine (Jat) ~21.5 264 nm; 345 nm Epiberberine (Epi) ~22.5267 nm; 357 nm Coptisine (Cop) ~23.5 264 nm; 358 nm Palmatine (Pal) ~26272 nm; 345 nm Berberine (Ber) ~27 263 nm; 347 nm4. Ginseng

(a) HPLC Conditions

-   -   Guard column: Lichrospher RP-18 endcapped        -   (5 μm, 4.0 ID×10 mm, Merck, German)    -   Column: Cosmosil 5C18-MS        -   (5 μm, 4.6 ID×250 mm, Nacalai tesque, Japan)    -   Column temperature: 35° C.    -   Mobile phase: A: 20 mM KH₂PO₄        -   B: CH₃CN        -   C: H₂O    -   Elution Gradients:

Time (minutes) A (%) B (%) C (%) Linearity 0 80 20 0 * 20 75 25 0 Linear40 65 35 0 Linear 55 0 80 20 Linear 60 0 20 80 Linear 65 80 20 0 Linear

-   -   Flow rate: 1.0 mL/min    -   Detection wavelength: 203 nm

(c) Results:

-   -   The HPLC chromatogram of the root of ginseng contains the        indicative ingredients of ginsenoside R_(g1), ginsenoside Re,        and ginsenoside R_(b1). The retention times and maximum        absorption wavelengths of these ingredients are shown in Table        5.

TABLE 5 Retention Times and Wavelengths of the Ingredients in GinsengMaximum absorption Compound Retention Time (minutes) wavelength (λmax)Ginsenoside R_(g1) (R_(g1)) ~23.5 204 nm Ginsenoside Re (Re) ~23.8 203nm Ginsenoside R_(b1) (R_(b1)) ~38.5 203 nm5. Ginger

(a) HPLC Conditions

-   -   Guard column: Lichrospher RP-18 endcapped        -   (5 μm, 4.0 ID×10 mm, Merck, German)    -   Column: Cosmosil 5C₈-MS        -   (5 μm, 4.6 ID×250 mm, Nacalai tesque, Japan)    -   Column temperature: 35° C.    -   Mobile phase: A: 20 mM KH₂PO₄        -   B: CH₃CN        -   C: H₂O    -   Elution Gradients:

Time (minutes) A (%) B (%) C (%) Linearity 0 70 30 0 * 20 40 60 0 Linear30 0 80 20 Linear 35 0 30 70 Linear

-   -   Flow rate: 1.0 mL/min    -   Detection wavelength: 280 nm

(d) Results:

-   -   The HPLC chromatogram of the rhizome of ginger contains the        indicative ingredients of 6-gingerol, and 6-shogaol. The        retention times and maximum absorption wavelengths of these        ingredients are shown in Table 6.

TABLE 6 Retention Times and Wavelengths of the Ingredients in GingerRetention Time Maximum absorption Compound (minutes) wavelength (λmax)6-Gingerol (G) 17 230 nm; 285 nm 6-Shogaol (S) 26 230 nm; 285 nm

PHARMACEUTICAL COMPOSITIONS

The pharmaceutical compositions of the present invention is prepared bythe following procedures:

(1) Preparation of Herbal Extracts

Some of the herbs (e.g., scutellaria, coptis, and rhubarb) are preferredto be individually extracted by a solvent. The solvent can be water ororganic solvent that is pharmaceutically acceptable for extractionpurpose, or a mixture of water and the organic solvent. The preferredorganic solvent is alcohol. It is preferred that the root of scutellariaand the rhizome of coptis are extracted by water, and the root andrhizome of rhubarb is by alcohol, especially 95% of alcohol in water(v/v). The extracts are further filtered.

(2) Condensation of the Herbal Extracts

For the herbs that have been prepared by extraction, the individualherbal extracts, after filtration, are pooled together and condensedunder reduced pressure in a water bath (maintained at 50° C.) until anherbal paste is formed.

(3) Preparation of Dry Powders

All of the herbs used in the herbal pharmaceutical compositions can beused in the form of dry powders. However, for better therapeuticeffects, all herbs, except ginseng/American ginseng or ginger, arepreferred to be prepared by solvent extraction. As to ginseng/Americanginseng or ginger, the dry powders form of the herbs are prepared bycutting the herbs into small pieces, followed by grinding and dryingthem into powders. The dry powders of the herbs are then passed througha sieve to ensure that the size of the dry powders are within certainranges.

(4) Preparation of A Herbal Pharmaceutical Composition

The dry powders of the herbs are mixed in with the herbal paste as shownin (2), supra, to form an herbal mixture, which is further dried to formthe pharmaceutical composition. Additionally, the pharmaceuticalcomposition can be processed into tablet, bolus, powder, capsule, andgranule by means of formulation which is well-known to those ordinaryskill in the art, particularly in the pharmaceutical industry.Excipients, binders, carriers, fillers can be added to the herbalmixture.

The following examples are for illustrative purpose and are not intendedto limit the scope of the invention. Reasonable variations, such asthose understood by reasonable artisans, can be made herein withoutdeparting from the scope of the present invention.

EXAMPLE 1 Preparation of Herbal Pharmaceutical Composition 1

Herbal pharmaceutical composition 1 of the present invention wasprepared as follows:

(1) About 20 grams of each of the root of scutellaria, the rhizome ofcoptis, the root and rhizome of rhubarb, and ginseng, in the form of“Yin Pian” (meaning “drinking pieces”), which contained small thinslices of the herb that were ready for decoction use, were individuallymeasured.

(2) The herbs of (1) were individually ground in a grinder intoindividual powder forms.

(3) The individual powders of (2) were passed though a 120-mesh sieverespectively and the resultant powders were collected.

(4) The individual powders of (3) were mixed together until ahomogeneous mixture was obtained.

EXAMPLE 2 Preparation of Herbal Pharmaceutical Composition 2

The herbal pharmaceutical composition 2 of the present invention wasprepared as follows:

(1) About 20 grams of each of the root of scutellaria, the rhizome ofcoptis, and the root and rhizome of rhubarb, in the form of “Yin Pian”(meaning “drinking pieces”), which contained small thin slices of theherb that were ready for decoction use, were individually measured.

(2) The individually measured herbs of (1) were separately simmeredand/or boiled in about 20 volumes of water for about 60 minutes toproduce individual herbal extracts.

(3) The individually boiled herbal extracts were passed through a100-mesh sieve when the extracts were still hot; and the filteredextracts were individually collected.

(4) The individually filtered herbs of (3) were mixed together andcondensed under condensed pressure in a 50° C. water bath until a pastewas formed.

(5) About 20 grams of the dry powders of ginseng or American ginsengwere prepared according to (1) and (2) of Example 1, supra.

(6) The herbal paste of (4) and the dry powders of (5) were mixedtogether until a homogeneous paste was obtained.

EXAMPLE 3 Preparation of Herbal Pharmaceutical Composition 3 (STC)

The herbal pharmaceutical composition 3 of the present invention wasprepared as follows (NOTE: The herbal pharmaceutical composition 3 isalso known as STC, which is named after “SunTen Cardiovascular Drug”):

(1) About 20 grams of each of the root of scutellaria, the rhizome ofcoptis, and the root and rhizome of rhubarb, in the form of “Yin Pian”(meaning “drinking pieces”), which contained small thin slices of theherb that were ready for decoction use, were individaully meansured.

(2) The small pieces of scutellaria and coptis were individuallysimmered and/or boiled in about 20 volumes of water for about 60 minutesto produce the extracts of scutellaria and coptis, respectively.

(3) The extracts of scutallaria and coptis were separately passedthrough a 100-mesh sieve when the extracts were still hot; and thefiltered herbal extracts were individually collected.

(4) The filtered herbal extracts of (3) were mixed together andcondensed under reduced pressure in a 50° C. water bath until a pastewas formed.

(5) About 20 grams of root and rhizome of rhubarb, in the form of “YinPian” were meansured, and extracted under refluxing in about 20 volumesof alcohol: water (95:5, v/v) for 60 minutes to produce a rhubarbextract.

(6) The rhubarb extract of (6) was passed through a 100-mesh sieve; andthe filtered rhubarb extract was collected.

(7) The filtered rhubarb extract was condensed under reduced pressure ina 50° C. water bath until a paste was formed.

(8) About 20 grams of dry powders of ginseng or American ginseng wasprepared according to (1) and (2) of Example 1, supra.

(9) The dry powders of (8) were mixed with the pastes of (4) and (7)until a homogeneous paste was obtained.

EXAMPLE 4 Preparation of Herbal Composition 4

The herbal pharmaceutical composition 4 of the present invention wasprepared using the same procedures as Example 2, except that the root ofscutellaria, the rhizome of coptis, and the root and rhizome of rhubarbwere extracted in alcohol: water (50:50, v/v), rather than water.

EXAMPLE 5 Preparation of Herbal Pharmaceutical Composition 5

The herbal pharmaceutical composition 5 of the present invention wasprepared using the same procedures as in Example 2, except that the rootof scutellaria, the rhizome of coptis, and the root and rhizome ofrhubarb were extracted in alcohol: water (95:5, v/v).

EXAMPLE 6 Preparation of Herbal Pharmaceutical Composition 6

The herbal pharmaceutical composition 6 of the present invention wasprepared using the same procedures as in Example 1, except that gingerwas used to replace ginseng or American ginseng.

EXAMPLE 7 Preparation of Herbal Pharmaceutical Composition 7

The herbal pharmaceutical composition 7 of the present invention wasprepared using the same procedures as in Example 2, except that gingerwas used to replace ginseng or American ginseng.

EXAMPLE 8 Preparation of Herbal Pharmaceutical Composition 8

The herbal pharmaceutical composition 8 of the present invention wasprepared using the same procedures as in Example 4, except that gingerwas used to replace ginseng or American ginseng.

EXAMPLE 9 Preparation of the Herbal Pharmaceutical Composition 9

The herbal pharmaceutical composition 9 of the present invention wasprepared using the same procedures as in Example 4, except that gingerwas used to replace ginseng or American ginseng.

EXAMPLE 10 Preparation of Herbal Pharmaceutical Composition 10

The herbal pharmaceutical composition 10 of the present invention wasprepared using the same procedures as in Example 5, except that gingerwas used to replace ginseng or American ginseng.

COMPARATIVE EXAMPLE 1 Preparation of Modified San-Huang-Hsie-Hsin-Tang

San-Huang-Hsie-Hsin-Tang contains the root of scutellaria, the rhizomeof coptis, and the root and rhizome of rhubarb. The commerciallyavailable San-Huang-Hsie-Hsin-Tang is a decoction, which contains the“Yin-Pians” (small thin slices) of the root of scutellaria, the rhizomeof coptis, and the root and rhizome of rhubarb. When in use, the“Yin-Pians” were placed in a bowl and boiled water was added to the“Yin-Pians”—containing bowl for drink as a soup or a beverage.Alternatively, the “Yin-Pians” of San-Huang-Hsie-Hsin-Tang can be placedin a boiler to be cooked with water.

To properly compare the herbal pharmaceutical compositions of thepresent invention (i.e., Examples 1–10) with San-Huang-Hsie-Hsin-Tang,the three ingredients of the San-Huang-Hsie-Hsin-Tang were prepared thesame way as that in Example 2, i.e., by boiling the “Yin-Pians” of theroot of scutellaria, the rhizome of coptis, and the root and rhizome ofrhubarb individually in water. The extracts were filtered, combined andthen condensed into a paste.

COMPARATIVE EXAMPLE 2 Preparation of Modified San-Huang-Hsie-Hsin-Tang

To properly compare the herbal pharmaceutical compositions of thepresent invention (i.e., Examples 1–10) with San-Huang-Hsie-Hsin-Tang,the three ingredients of the San-Huang-Hsie-Hsin-Tang were prepared thesame way as that in Example 3, i.e., by boiling the “Yin-Pians” of theroot of scutellaria and the rhizome of coptis individually in water andextracting the root and rhizome of rhubarb in alcohol:water (95:1, v/v)under refluxing. The extracts of scutellaria and coptis were separatelyfiltered, combined, and condensed into a paste. The extract of rhubarbwas filtered and condensed into a paste. The paste of scutellaria andcoptis and the paste of rhubarb was then combined and mixed thoroughly.

Pharmacological Studies

The following pharmacological studies confirm that the herbalpharmaceutical compositions of Examples 1–10, supra, were capable oflowering high blood pressure, maintaining stable blood pressure (in thenormal range), improving cardiovascular diseases in the elderly,inhibiting the formation of nitrite, inhibiting the proliferation ofsmooth muscle, reducing CRP formation, inhibiting iNOS expression, andinhibiting COX-2 activity. Such herbal compositions were not only usefulfor treating patients with normal physique, but also safe and effectivefor treating patients who were elderly or debilitated.

Although herbal pharmaceutical compositions 1–10, supra, alldemonstrated therapeutic effectiveness in treating patients withcardiovascular diseases, Example 3, also known as STC, appeared to besuperior to the rest of the Examples. Therefore, for the purpose ofsimplifying the studies to be presented below, only STC was chosen asrepresenting the pharmaceutical composition of the present invention.

Study 1 Effects of the Herbal Compositions on Lowering Blood Pressure

Spontaneous hypertensive rats (SHR) were obtained from the NationalExperimental Animal Center, Taiwan. The SHR rats were firstanaesthetized by intraperitoneal injection of urethane at 9 g/kg. TheSHR rats were then inserted with a trachea tube, maintained on arespiration apparatus for small animals, and inserted with cathetersinto the femoral vein and femoral artery.

The catheter from the femoral artery was connected to a pressuretransducer, which transmitted the change in pressure to a multi-functionrecorder for monitoring of the artery blood pressure, mean arterialblood pressure, and heart rate. The femoral vein catheter was in placefor intravenous injection. The body temperature of the rats wasmaintained at 37–38° C. by an electric blanket. The baseline values wererecorded after the blood pressure of the SHR rats were stabilized.

The SHR rats were separated into six groups and received a single doseof one of the following medications through gavage: (1) 144 mg/kg of theroot of scutellaria alone (H1); (2) 144 mg/kg of the root and rhizome ofrhubarb alone (H3); (3) 144 mg/kg of the combination of the root ofscutellaria and the rhizome of coptis (H1+2); (4) 144 mg/kg of thecompositions San-Huang-Hsie-Hsin-Tang as in COMPARATIVE EXAMPLE 1 (M);(5) 144 mg/kg of STC (Example 3); and (6) the combination of captopril(ACE inhibitor) 1 mg/kg and nifedipine (calcium channel blocker) 0.4mg/kg (A). The SHR rats in the Western medicine control group (A) hadbeen pre-treated with one week of captopril and nifedipine at the samedoses.

Blood pressure and heart rate were monitored continuously throughout thestudy period. Percent change in the mean arterial blood pressure (maBP)was expressed as the ratio of maBP after treatment to the baseline maBPbefore treatment. The within-group baseline and post-treatment resultswere analyzed using the paired Student's T test.

As presented in FIG. 1, all medications showed significant reduction ofthe blood pressure (indicated with an asterisk,*). However, the root andrhizome of rhubarb alone (H3) was less effective in lowering the bloodpressure. Also, the combination of the root of scutellaria and therhizome of coptis (H 1+2) show a large variation in the blood pressurereduction.

Study 2 Effects of the Herbal Compositions on Maintaining andControlling Blood Pressure

Spontaneous hypertensive rats (SHR) were obtained from the NationalExperimental Animal Center, Taiwan. The SHR rats were anaesthetized byintraperitoneal injection of urethane at 9 g/kg. The SHR rats were theninserted with a trachea tube, maintained on a respiration apparatus forsmall animals, and inserted with catheters into the femoral vein andfemoral artery.

The catheter from the femoral artery was connected to a pressuretransducer, which transmitted the change in pressure to a multi-functionrecorder for monitoring of the artery blood pressure, mean arterialblood pressure, and heart rate. The femoral vein catheter was in placefor intravenous injection. The body temperature of the rats wasmaintained at 37–38° C. by an electric blanket. The baseline values wererecorded for 60 minutes after the blood pressure of the SHR rats werestabilized.

The SHR rats were separated into groups (seven rats per group) andreceived through gavage daily for two weeks of (1) no treatment(control); (2) 48 mg/kg of composition of COMPARATIVE EXAMPLE 2 (M); (3)48 mg/kg of STC (Example 3); or (4) the combination of captopril 1 mg/kgand nifedipine 0.4 mg/kg (A). The dosages of M and STC were one-third ofthose of STUDY 1.

Blood pressure and heart rate were monitored continuously throughout thestudy period. Values of the mean arterial blood pressure (maBP) andheart rate (HR) were compared on the day after the completion of the2-week dosing. The between-group results were analyzed using theunpaired Student's T test. The within-group, baseline and post-treatmentresults were analyzed using paired Student's T test.

As shown in FIG. 2 a, only the group giving STC demonstrated lower andmaintained mean arterial blood pressure on the day after the completionof the doses. In contrast, the traditional San-Huang-Hsie-Hsin-Tangdecoction (M) and the combination of captopril and nifedipine (A) didnot maintain their anti-hypertensive effects on the day after dosecompletion, despite the multiple dose administration for two weeks.

As shown in FIG. 2 b, all three treatments had no effects on the heartrate.

Study 3 Effect of the Herbal Compositions on Preventing Hypertension

The spontaneous hypertension rats (SHR) and normal rats (WKY) youngerthan 8 weeks old were obtained from the National Experimental AnimalCenter, Taiwan. Baseline measures of heart rate and blood pressures atthe tail are first recorded. The SHR rats were then treated with 14.4mg/kg/day of the herbal compositions of the present invention asdescribed in the below Example 4 through gavage for one week. Thebetween-group comparison of the baseline measures of the WKY and SHRrats was carried out using the unpaired Student's T test with thestatistical significance indicated by an asterisk in the figures. Thewithin-group comparison of the baseline and post-treatment measures ofthe SHR rats was carried out using paired Student's T test with thestatistical significance indicated by an “a” in the figures.

As shown in FIGS. 3 a and 3 b, the untreated spontaneous hypertensiverats had statistically significantly higher heart rate (HR), systolicpressure (SYS), diastolic pressure (DIA) and mean arterial bloodpressure (MED) than the normal rats (WKY). Treatment with the herbalcompositions of the present invention (STC) significantly reduced thesystolic pressure and mean arterial blood pressure in the spontaneoushypertensive rats (p<0.05, n=5) to levels that were comparable to thenormal WKY rats.

Study 4 Effect of the Herbal Compositions on Improving CardiovascularConditions in the Elderly

Part 1. In Vivo Test

Normal WKY rats that were 8 weeks and 10 months of age were used in thisstudy. Baseline blood pressures and heart rate measured at the tailswere first recorded for both groups. The 10-month old rats were thentreated with one single dose of 48 mg/kg of the herbal compositions ofthe present invention. Heart rate and blood pressures were measured at 1hour after the dosing of STC. The between-group comparison of thebaseline measures of the 8-week old (Young) and the 10-month old WKYrats (Old) was carried out using the unpaired Student's T test with thestatistical significance indicated by an asterisk in the figures. Thewithin-group comparison of the baseline (Old) and post-treatmentmeasures of the 10-week old WKY rats (STC) was carried out using pairedStudent's T test with the statistical significance indicated by an “a”in the figures.

As shown in FIGS. 4 a and 4 b, the heart rate (HR), diastolic pressure(DIA) and mean arterial blood pressure (maBP) were higher, but notsignificantly, in the old WKY rats than the young rats. Treatment withthe herbal compositions of the present invention (STC) significantlyreduce the diastolic pressure and mean arterial blood pressure in theold WKY rats (p<0.05, n=4, indicated with “a” in FIG. 4 b) at one hourafter the dose administration.

Part 2. Non-Circulation Perfusion Test

Ten-month old SHR rats were anaesthetized by intraperitoneal injectionof urethane. The hearts of the anaesthetized rats were removed underassisted respiration. The hearts were immediately inserted with anarterial catheter at the cutting end of the aorta and perfused with 37°C. Krebs-Henselein (KH) buffer at the pressure of 80 cmH₂O. The buffercontained 118 mM NaCl, 24.0 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mMMgSO₄, 1.7 mM CaCl₂, and 10.0 mM glucose continuously gassed with themixture of 95% O₂ and 5% CO₂.

The isolated hearts were perfused for 15 minutes with the KH buffer andthen with the KH buffer containing the herbal compositions of thepresent invention (STC) for 15 minutes, reaching a steady state. Thecoronary flow and coronary perfusion pressure (CPP) were measured (1)before the perfusion of STC, (2) after the perfusion of STC, and (3)after reperfusion with STC for 60 minutes post L-phenylephrine-inducedischemia. The results indicated that perfusion with the herbalcompositions of the present invention as described in the below Example4 could improve the reduction in the coronary flow that was induced by10 mM L-phenylephrine from 12.6 mL/min to 13.2 mL/min. This suggestedthe herbal pharmaceutical compositions of the present invention waseffective in improving cardiovascular conditions.

Study 5 Effects of the Herbal Compositions in Inhibiting the EnzymeFormations and Activities of iNOS and COX-2

The mouse macrophage cell line RAW 264.7 was used in this study. Themouse macrophage cells were treated with lipopolysaccharide (LPS) andvarious amounts of the herbal compositions of the present invention for12 hours. LPS is known to induce the syntheses of the enzymes, iNOS andCOX-2 in the mouse macrophage cells. The treated macrophage cells werefirst detached from the cultural plates by repeated flush usingmicropipett. The cell mixtures were then harvested and centrifuged at2000 rpm. The clear supernatants were collected and assayed for thecontent of nitrile using the Griess Test Reagent and for the content ofPGE₂ using the ELISA method. The pellets formed after the centrifugationwere mixed with the Lysis buffer and homogenized. The resultant mixtureswere assayed for protein content. A 30-μg aliquot of the homogenizedmixture was placed on the polyacrylamide gel to separate the proteins byelectrophoresis. Proteins on the developed polyacrylamide gel were thentransferred onto the polyvinylidene difluoride (PVDF) membrane anddetected with the antibodies for iNOS and COX-2.

The results showed that the herbal compositions of the present inventionwas effective in inhibiting the formation of the nitrile (FIG. 5) byinhibiting the protein synthesis of iNOS. The herbal compositions of thepresent invention inhibited the formation of PEG₂, the end product ofCOX-2 (FIG. 6), but not the protein synthesis of the enzyme itself,suggesting that the herbal compositions of the present invention mightpossess anti-inflammatory activity by inhibiting the activity of COX-2.

Study 6 Effect of the Herbal Compositions on Inhibition of iNOS andCOX-2 Protein Biosynthesis

Sprague Dawley (SD) rats and the spontaneous hypertensive (SHR) ratswere used in this study. The hearts from lipopolysaccharide-treated SDrats and the lungs from SHR rats treated with the herbal compositions ofthe present invention and other control groups were removed from theanimals. Small portions of the isolated organs were first ground inmortars containing liquid nitrogen and then homogenized in the Lysisbuffer.

Aliquots of the resultant mixtures were assayed for protein contents.Another 30-μg aliquots of the resultant mixtures were placed on thepolyacrylamide gel to separate the proteins by electrophoresis. Proteinson the developed polyacrylamide gel were then transferred onto thepolyvinylidene difluoride (PVDF) membrane and detected with theantibodies for iNOS and COX-2.

The results showed that intraperitoneal treatment of 20 mg/kg LPSinduced inflammatory responses in the SD rats. The SD rats showedsymptoms of abdominal pain and writhing. As shown in FIGS. 7 a and 7 b,formations of the COX-2 and iNOS proteins, respectively, were highest inthe hearts of the SD rats (Column 1), then followed by the lungs of theuntreated SHR rats (Column 3). STC (Example 3) clearly inhibited theformation of the COX-2 and iNOS proteins in the SHR rats (Column 2).

Study 7 Effect of the Herbal Compositions on Inhibition of iNOS GeneExpression

The spontaneous hypertensive (SHR) rats were treated with (1) thepharmaceutical compositions of the present invention, STC (S); (2)western medicine; or (3) control. Lungs from the animals were removedand tissue RNA was extracted using Trizol. The concentrations and ratiosof the extracted RNA were determined using the spectrometer. The cDNAwas then prepared from 1-μg aliquot of RNA by reverse transcription. A2-μL of the cDNA solutions was mixed with the iNOS primer and multipliedusing the polymerase chain reaction (PCR) method. The resultant DNAmixtures were run through the agarose gel for DNA separations.

As indicated in 8a, detection of the β-actin cDNA on the agarose gelindicated the RNA extraction process was properly carried out. As shownin FIG. 8 b, the DNA bands was clearly seen in the western medicinegroup (A), less obvious in the control (untreated) group (C) and notobserved for the SHR rats treated with the herbal compositions of thepresent invention (S). This suggested that the herbal compositions ofthe present invention could inhibit the expression of iNOS in SHR rats.

Study 8 Effect of the Herbal Compositions on Reduction of CRP

The spontaneous hypertensive rats were treated with 48 mg/kg of STC(Example 3) for 2 weeks. After the conclusion of the oral doseadministration, blood samples were collected from both the control(untreated) rats and the STC-treated rats. Plasma samples were preparedfrom the blood samples by high-speed centrifugation and then assayed forthe CRP contents using ELISA.

As indicated in FIG. 9, the herbal pharmaceutical compositions of thepresent invention was effective in reducing CRP formation in thespontaneous hypertensive rats.

While the invention has been described by way of examples and in termsof the preferred embodiments, it is to be understood that the inventionis not limited to the disclosed embodiments. On the contrary, it isintended to cover various modifications as would be apparent to thoseskilled in the art. Therefore, the scope of the appended claims shouldbe accorded the broadest interpretation so as to encompass all suchmodifications.

1. A method for treating patients with hypertension and/or ischemia comprising: administering an effective amount of an herbal pharmaceutical composition to said patients; wherein said herbal pharmaceutical composition comprising: an extract of Radix Scutellariae (root of scutellaria); an extract of Rhizoma Coptidis (rhizome of coptis); an extract of Radix et Rhizoma Rhei (root and rhizome of rhubarb); and dry powder of Radix Ginseng (root of ginseng) or Radix Panacis Quinquefolii (American ginseng); wherein said extract of said root of scutellaria, said extract of said rhizoma of coptis, and said extract of said root and rhizome of rhubarb are produced by extracting said root of scutellaria, said rhizoma of coptis, said root and rhizome of rhubarb by a solvent which is at least one selected from the group consisting of water and alcohol.
 2. The method according to claim 1, wherein said herbal pharmaceutical composition is orally administered to patients.
 3. The method according to claim 1, wherein said herbal pharmaceutical composition is used to lower blood pressure or maintain stable blood pressure in said patients. 